Toll-like receptors and integrins crosstalk

Immune system recognizes invading microbes at both pathogen and antigen levels. Toll-like receptors (TLRs) play a key role in the first-line defense against pathogens. Major functions of TLRs include cytokine and chemokine production. TLRs share common downstream signaling pathways with other receptors. The crosstalk revolving around TLRs is rather significant and complex, underscoring the intricate nature of immune system. The profiles of produced cytokines and chemokines via TLRs can be affected by other receptors. Integrins are critical heterodimeric adhesion molecules expressed on many different cells. There are studies describing synergetic or inhibitory interplay between TLRs and integrins. Thus, we reviewed the crosstalk between TLRs and integrins. Understanding the nature of the crosstalk could allow us to modulate TLR functions via integrins.


Introduction
Immune cells are mounted with a number of pattern recognition receptors (PRRs) that recognize foreign pathogens.Microbial components are main targets for host immune cells to use for the recognition of microbes, and Toll-like receptors (TLRs) are one of major PRRs and evolutionarily ancient mediators for innate host defense (1,2).Other PRRs include RIG-I-like receptors (RLRs), Nod-like receptors (NLRs), and C-type lectin receptors (CLRs) (3).So far 10 human TLRs (TLR1-TLR10) and 12 mouse TLRs (TLR1-9, TLR11-13) are identified (4).They are expressed on the plasma membrane or the endocytic vesicles.
TLR9 recognizes bacterial and viral CpG DNA motifs (15,16).The recognition of microbial pathogens by TLRs induces the activation of intracellular signaling pathways, resulting in the production of inflammatory cytokines, type I interferon, and chemokines.TLRs also induce the upregulation of costimulatory molecules on dendritic cells (DCs) (17).TLR10 is the latest human TLR to be discovered, and its ligand is still unclear (18).In contrast to TLR1-9, TLR10 demonstrates anti-inflammatory response (19,20).While it is known to respond to influenza virus infection (21), this TLR still requires more extensive work in the future.
In addition to recognizing exogenous ligands derived from microbes, TLRs interact with endogenous molecules released from damaged tissues or dead cells (22).For example, high mobility group box 1 (HMGB1) is a nonhistone nuclear protein (23) and can bind to TLR2, TLR4, and TL9.The list of ligands for each TLR is listed in Table 1.The location of each TLR is also shown.
The production of pro-inflammatory mediators such as cytokines and chemokines is one of the major TLR functions.Those inflammatory mediators would help regulating the immune system (35).For example, TLR2 and TLR4 are recognized by various ligands (Table 1).However, inflammatory response by different TLR2 ligands may not be the same.The involvement of non TLR receptors can provide a more tailored, specific response to TLRs.Integrins are critical adhesion molecules involved in many biological processes and play an important role in TLR crosstalk.Thus, we will first describe TLR signaling pathways.Then we will examine the role of integrins as regulators of TLR functions.

MyD88 signaling pathway
TIR domains are essential components of the innate immune system (36).The proximal events of TLR-mediated intracellular signaling are initiated by the interaction of TIR-domain of TLRs with TIR-domain-containing cytosolic adaptors and MyD88 is a central adaptor protein for TLRs.With the exception of TLR3, all TLRs mediate the downstream signaling pathway via MyD88 (37).The association of TLRs with MyD88 recruits the members of the interleukin-1 receptor associated kinase (IRAK) family, forming MyD88-IRAK-4 complex.This recruits IRAK-1 and IRAK-2, leading to the phosphorylation of IRAKs and interaction with tumor necrosis factor receptor associated factor 6 (TRAF6).TRAF6 induces the activation of transforming growth factor-b activated kinase 1 (TAK-1), thereby I-kB (IkB) and mitogenactivated protein kinase (MAPK).The activation of IkB and MAPK results in nuclear factor kappa B (NF-kB) and activator protein 1 (AP-1)-mediated gene transcription (38,39).IRAK activation also stimulates interferon-regulatory factor (IRF) such as IRF7 (40)(41)(42) and activates the gene transcription of type I IFN (43).As a result, pro-inflammatory cytokines including tumor necrosis factor (TNF), interleukin (IL)-1, IL-6, IL-12, and interferon (IFN)-a are produced (44).

Integrins
Integrins are a/b heterodimeric cell adhesion molecules that mediate cell-to-cell and cell-to-matrix interactions (47, 48).They are type I membrane glycoproteins with large extracellular domains, single transmembrane domains, and relatively short intracellular tails.The head of the large extracellular domain serves for ligand binding.To date, 18 aand 8 b-subunits have been identified that combine to form at least 24 distinct a/b heterodimers.The list of integrins with representative ligands is included in Table 2. Integrins on the membrane (the outside) can receive signals triggered by non-integrin receptors via intracellular signaling (inside-out signaling) and vice versa (outside-in signaling) (Figure 2) (52).

Inside-out signal
Inside-out signal is initiated by non-integrin receptors such as G protein-coupled receptors (GPCRs), selectins, and chemokine  56).The activation of T cell receptor (TCR) and tyrosine kinase Lck leads to the phosphorylation of ZAP-70 kinase.This triggers the phosphorylation of LAT adaptor protein and the activation of phospholipase Cg1.This ultimately activates the small G protein ras-related protein-1 (Rap1).Rap1 binds to Rap-1 interacting adaptor molecule (RIAM).These events trigger the binding of talin and kindlin to b2 subunit, which induces the conformational change of aLb2 into its active form (57).Although the binding of talin alone to integrin can activate it, its potency is extremely weak, supporting the critical role of kindlin in this process (58).The activation of aLb2 results in its binding with ligands including intercellular adhesion molecule-1 (ICAM-1).What kind of molecules each integrin uses for inside-out signal and whether the same integrin uses different combination of molecules depending on cell type would be an important research area.

Outside-in signal
Upon the inside-out activation, an integrin binds to a specific ligand.However, for the integrin to tightly bind to its ligand to mediate cell adhesion and migration, its cytoplasmic domains must be anchored to the cytoskeleton (59,60).When the integrin binds to its ligand, it triggers the assembly of large protein complexes known as focal adhesions by incorporating a variety of molecules including cytoskeletal proteins and signaling molecules.Linking the integrin to the actin cytoskeleton promotes firm cell adhesion, cell spreading, migration and proliferation (57).Talin and kindlin serve as seed proteins to recruit proteins and initiate focal adhesion assembly (61).In case of aLb2, the binding of ICAM-1 induces the activation/deactivation of kinases and phosphatases, leading to the cytoskeletal remodeling for the fine-tuning of effector functions such as T cell migration (62).Interestingly, this outside-in signal can be modified by the heterotrimeric guanine nucleotidebinding protein (G protein) Ga13.GPCRs activate Ga13, triggering its interaction with b integrin to regulate the outside-in signal (53).

Integrin-TLR crosstalk b1 integrin
b1 integrin receptors regulate numerous functions, including cell adhesion, migration, differentiation, growth, and survival.b1 integrin subfamily consists of 12 a-chains that non-covalently bind to b1 chain (CD29) (49,63).They can be categorized by their binding characteristics such as Arg-Gly-Asp (RGD)-binding integrins (avb1, a8b1, and a5b1), Leu-Asp-Val (LDV)-binding integrins (a4b1 and a9b1), collagen-binding integrins through triple helical GFOGER sequence in major collagens (a1b1, a2b1, a10b1, and a11b1), and laminin-binding integrins which includes both non-a Inserted (I) domain-containing integrins (a3b1, a6b1 and a7b1) and aI domain-containing integrins (a10b1, a2b1, and a1b1) (64).The key downstream signaling molecules of integrins include focal adhesion kinase (FAK), AKT, MAPK, Src-family protein tyrosine kinases, and integrin-linked kinase (ILK) (65).Integrins regulate intracellular signal transduction cascades that control differentiation, proliferation, and survival.Upon binding to fibronectin, collagen, and laminin, b1 integrin induces cell adhesion and migration that is extensively observed in pancreatic cancer models.Blockade or knockdown of b1 on cancer cells resulted better prognosis by reducing tumor growth and metastasis (66), which makes b1 integrin as an attractive therapeutic target.b1 integrins, in particular a9b1 has been reported to induce Th17 cell promoting cytokines in dendritic cells and macrophages in synergy with TLR2 and TLR4 through ERK pathway, that developed functional Th17 cells and arthritis (67).In addition to that, upon engaging with extracellular matrix (ECM) or other ligands, they initiate signaling pathways that can either reinforce or inhibit the activity of other receptors through negative or positive feedback loops.Interactions of a3b1 and a4b1 with TLRs have reported in several studies, which will be discussed in the following sections.

i. a3b1
a3b1 is expressed extensively on nearly all types of cells.It binds to a wide range of ligands with or without classical RGD integrinbinding motifs (68).a3b1 integrin serves as a receptor for collagen (type I and VI), laminin (a 1 b 1 g 1 ), laminin-5 (a 3 b 3 g 2 ), laminin-10 (a 5 b 1 g 1 ), laminin-11 (a 5 b 2 g 1 ), fibronectin, entactin, nidegon, and thrombospondin-1 with high specificities and affinities (69).Integrins are often targeted by bacterial and viral pathogens to adhere to and invade host cells.b1 integrins are particularly prone to their targets (70).b1 integrins serve as receptors for bacterial surface proteins including invasin and FimH (71, 72).a3b1 binds to BBB07 expressed on Borrelia burgdorferi (B.burgdorferi), the causative microbe of Lyme disease (73).BBB07 also serves as a TLR2 ligand.By ligation to the same ligand by both a3b1 and TLR2/1, human macrophages manifested enhanced proinflammatory responses to bacterial components.
a3b1 also mediates the endocytosis of TLR2 ligand Pam 3 CSK 4 , thereby facilitating its recognition by TLR2/1 within the endosome (74).This leads to the recruitment of adaptor molecules such as MyD88 by TLR2/1, eventually activating NF-kB signaling pathway and inducing the production of pro-inflammatory cytokines such as IL-6 (56).In murine macrophages, the endosomal activation of TLR2/1 induces IFN-b (75).This endocytosis mediated by a3b1 was observed for both live bacteria and bacterial proteins.The impact on TLR2 mediated signaling via a3b1 is cell typedependent (76).As in macrophages, a3b1 and TLR2/1 crosstalk selectively enhances IL-6 and IL-10 production by neutrophils in the setting of sepsis.However, neutrophils do not produce TNF production.Activated neutrophils release laminin (77) which bind to a3b1 on their cell surface, and increase the phosphorylation of FAK, but not Syk.This is responsible for the aforementioned profiles of pro-inflammatory cytokines by neutrophils (78).Activated FAK feeds into the MyD88-dependent TLR signaling.It is not certain about the presence of direct interaction between a3b1 and TLR2/1 on the neutrophils, but it is suggested that they may interact transiently within the lipid rafts upon activation since both of them localize there during activation (79,80).
ii. a4b1 a4b1, also referred to as very late antigen-4 (VLA-4), is expressed on most leukocytes.It plays a crucial role in cell homing, trafficking, differentiation, activation, and survival.The ligands of this receptor include ECM protein fibronectin and the vascular cell adhesion molecule-1 (VCAM-1), which are expressed on endothelial cells (81).a4b1 binding site to fibronectin contains the tripeptide sequence Leu-Asp-Val (LDV) and is located in the alternatively spliced connecting segment 1 (CS-1) region, while VCAM-1 is recognized through the sequence Ile-Asp-Ser (IDS) (82).The domain called extra domain A (EDA) within fibronectin activates TLR4 (83).Thus, fibronectin severs as a ligand for both a4b1 and TLR4 (84).a4b1 was shown to function as a co-receptor for TLR4 in fibroblasts.Blockade of a4b1 or TLR4 or knockdown of a4 subunit in fibroblasts resulted in a decreased production of proinflammatory cytokines such as TNF and IL-10 (85).
b2 integrin b2 integrins consist of four members-aLb2 (CD11a/CD18, lymphocyte function-associated antigen-1), aMb2 (CD11b/CD18, macrophage-1 antigen, complement receptor 3), aXb2 (CD11c/ CD18, p150.95,complement receptor 4), and aDb2 (CD11d/ CD18).aLb2 is ubiquitously expressed on all leukocytes, while aMb2, aXb2, and aDb2 are mainly expressed on myeloid cells at different levels (86).aLb2 binds to intercellular adhesion molecule (ICAM)-1~5 that can be found on the surface of other cells.aMb2 has broad versatility, having over 40 known binding partners, such as ICAMs, iC3b, fibrinogen, RAGE (receptor for advanced glycation end products), and CD40L (87).aMb2 and aXb2 share several ligands as including iC3b, ICAM-1 and fibrinogen, but their binding sites on the same ligand are not exactly the same (88).aDb2 also binds to multiple ligands, encompassing extracellular matrix-associated proteins like fibronectin, fibrinogen, vitronectin, and plasminogen as well as ICAM-1 (89).Reactive oxygen species (ROS) produced by the ligation of TLR2 and TLR5 induced rapid b2-integrin activation on myelomonocytes, and promoted leukocyte adhesion, suggesting that TLRs collaborate with one another (90).CD18 (b2) knockout (KO) macrophages and DCs produced higher level of IL-12p40 and IL-6 in response to TLR2, TLR4 and TLR9 stimulation, and higher level of type I interferon in Integrin signaling Inside-out signal: Integrins are in an inactive conformation at baseline.However, the activation of receptors such as GPCRs, chemokine receptors, and TCR induces a cascade of events within the cells.The example shown here is via TCR.At the end, talin along with kindlin bind to b subunit of integrins, inducing its conformational change, which triggers the structural change of a subunit, allowing the integrin to bind to its ligand.Outside-in signal: Integrins that bind to their ligands cause cytoskeletal changes via focal adhesion molecules including focal adhesion kinase (FAK), leading to cell proliferation, survival, differentiation, and migration.
response to TLR4 stimulation (91), suggesting that b2 integrins modulate TLR response.Further investigation of b2 ablation showed NF-kB and p38 MAPK pathway activations were involved in these processes (91,92).Among b2 integrins, the interplay between aMb2 and TLRs is well studied, which will be discussed further.
i. aMb2 aMb2 is highly expressed on macrophages, DCs, monocytes, granulocytes, and mature or activated NK cells (93).It regulates TLR signaling positively or negatively, depending on cell types and inflammatory status.
TLR4 KO neutrophils reduced aMb2 activation, but not aLb2 or aXb2, suggesting that TLR4 would selectively facilitate the activation of aMb2 on neutrophils.TLR4-mediated aMb2 induction involved the activation of transcription factors NF-kB and c-Jun (94).aMb2 can affect several TLRs.Upon in vivo challenge with TLR ligand stimulations (LPS, poly(I:C), and CpG) pro-inflammatory cytokines (TNF, IL-6, IL-10, and IFN-b) were greatly increased in the serum of CD11b (aM) KO mice (39).Higher level of pro-inflammatory cytokines in the serum was observed in CD11b KO mice during methicillin-resistant Staphylococcus aureus (MRSA) (95) and Escherichia coli (E.coli) <i> (96) </i>infection.Bacterial loads were higher in CD11b KO mice following MRSA and E. coli infections.In contrast, CD11b KO mice demonstrated better clearance of L. monocytogenes following its infection, despite higher serum TNF and IL-6 levels were detected (95).The difference in the phenotype may be because TNF induces apoptosis of certain bacteria (97).In case of MRSA and E.coli infection, TLR4 ligation activated aMb2 on macrophages by inside-out signaling through PI3K and RapL pathway, which negatively looped back TLR4 signaling (Figure 3) (96).Outside-in signaling activated Src-Syk and promoted degradation of MyD88 and TRIF (Figure 3).This feedback loop in macrophages may control balance of both TLR4 and aMb2 signaling pathways since their uncontrolled activation can cause harmful pathogenesis.Of note, syk is typically associated with other receptors such as C-type lectin receptors (CLRs).To make complicated further, resident macrophages or bone marrow derived macrophages from CD11b KO mice showed similar level of pro-inflammatory cytokines and activation status upon LPS stimulation, thus suggesting that the interplay of aMb2 with TLR4 was not involved in steady-state macrophages (96).Thus, the interplay between TLRs and aMb2 may be dictated by cell types and their cellular state.In fact, the lack of aMb2 in DCs resulted in decreased pro-inflammatory cytokines and reduced MyD88-dependent phosphorylation of p38, Erk1/2, JNK, and IkBa in response to LPS stimulation (96).Upon stimulation with LPS, aMb2 was clustered in DCs and colocalized with CD14, which has been shown important for TLR4 endocytosis, suggesting that aMb2 was a part of TLR4 endocytosis.Furthermore, CD11b KO in DCs impaired RANTES production in LPS induced TRIF-mediated signaling in the endosome (44).Unlike TLR4, aMb2 in DCs negatively regulated TLR9 signaling by selectively reducing IL-12p70 production, which was possibly regulated by upregulated miR-146.The consequence of IL-12p70 production affected poor cross-priming of DCs to cytotoxic T lymphocyte (CTL) response (98).TLR3 and aMb2 interplay has been reported on NK cells.KO and neutralization of aMb2 enhanced cytotoxic function of NK cells in response to TLR3 stimulation and limited acute liver infection (93).aMb2 deficiency impaired the activation of MAPK/JNK pathway, suggesting that it inhibited TLR3 mediated activation of NK cells (99).Inside-out activation of aMb2 by TLR2 in association with CD14 was reported in monocytes during the infection of Porphyromonas gingivalis, a pathogen implicated in chronic periodontitis and atherosclerosis.The activation of aMb2 induced adhesion and recruitment of monocytes to the site of the infection (100).This recruited inflammatory monocytes can be beneficial to control infection, but uncontrolled accumulation results a tissue destruction.Although current data are all based on either aMb2 or TLR KO system or depletion by neutralizing antibodies, the studies suggested a possible indirect interplay between aMb2 and TLR2 (100).
While most studies examined the interaction between TLRs and aMb2 by inhibiting or deleting aMb2, some studied by activating it.aMb2 activation by leukadherin-1 (LA1), its allosteric agonist, protected mice from pathological injuries and reduced the mortality induced by LPS (101).aMb2 activation by LA-1 inhibited M1 macrophage response to LPS both in vivo and in vitro.Although it is not clear whether LA-1 facilitated a direct interaction between aMb2 and TLR4 on macrophages, it induced an endocytosis of both aMb2 and TLR4 and prevented LPS binding to TLR4 (101).While it prevented an excessive activation of TLR4 signaling pathway and pro-inflammatory response in macrophages, LA-1 pretreatment induced pro-inflammatory cytokines in DCs, suggesting that the effect of LA-1 could be cell type-dependent (101).It is worth noting that the expression levels of aMb2 on macrophages and DCs are different (102), which may be in part responsible for the different effect of LA-1 on these two cell types.
A recent study showed that CD11b deficiency of donor nonclassical monocytes increased CXCL2 production and exacerbated primary graft dysfunction in lung transplantation model (103).High mobility group box 1 (HMGB1), a DAMP released from dying cells, activates TLR4 in nonclassical monocytes.It was released from the donor lungs with primary graft dysfunction.Interestingly, HMGB1 stimulation induced lower CXCR2 production by TLR4 single KO or TLR2/TLR4 double KO monocytes, but not TLR2 single KO.It is uncertain whether aMb2 interacts directly with TLR2 or TLR4, however, aMb2 agonist LA-1 prevented primary graft dysfunction, suggesting that aMb2 might facilitate TLR endocytosis.
Although all the evidence supports the presence of crosstalk between aMb2 and TLRs, several questions still need to be answered.Do aMb2 and TLRs bind to the same ligands?In fact, several studies reported that aMb2 binds viral dsRNA (104), and bacterial toxins (105) and LPS (106-108).So far the interaction between ligands and aMb2 has been shown in vitro.Therefore, it will be critical to determine if reported ligands for aMb2 are in fact relevant in vivo.If so, it is not known whether both TLRs and aMb2 bind a ligand at the same time.If they do, which signaling should be activated first?Does ligand binding avidity and affinity affect downstream signal?Or do they limit activation?If they don't, is there ligand binding competition between TLR and aMb2?Would it be possible that the rest of b2 integrin members interplay with TLRs?This may be very possible since extracellular part of aXb2 has over 80% of sequence homology to aMb2 and about 50% of homology in intracellular tail, for example (109,110).Furthermore, LPS also binds to caspase-11 intracellularly (111), which makes the crosstalk complicated.b2 integrin members may also collaborate each other to inhibit or induce TLR response, since b2 integrin members are expressed upon activation of cells, especially on myeloid cells.They may function synergistically.Apparently, the proposed crosstalk between aMb2 and TLRs depend on cell types, but does ligand binding affinity or avidity affect crosstalk?For example, high-affinity ligand binding affects the degree of up-or downstream signal?There might be a rivalry between the ligands.It is interesting to know whether these crosstalks depend on the timing of activation or not.A previous study showed aMb2 on dendritic cells was activated through inside-out signaling by TLR4 (112) that was necessary for aMb2-induced phagocytosis but not affected aXb2, suggesting a bidirectional action between aMb2 and TLR4.
aV integrin aV integrin also known as CD51 or MSK8, is a transmembrane protein that is involved in cell adhesion, migration, and signaling (113).aV integrin forms heterodimers with various b integrin subunits such as b1, b3, b5, b6, and b8.Together they designate a various array of receptors to bind to specific ligands in the extracellular matrix (ECM) including fibronectin, vitronectin, fibrinogen, and osteopontin, enabling cells to adhere and respond to their surrounding environment (114,115).
In addition to adhesion, aV integrin promotes the activation of a multitude of signaling pathways, primarily the FAK pathway (116).The phosphorylation of FAK will in turn recruit Src kinases, phosphoinositide 3-kinase (PI3K) subunit p85, or phospholipase (PL)Cg and stimulate the signaling cascades of Ras/Erk, PI3k/Akt, and Crk/Dock180/Rac.These pathways contribute to cell survival, proliferation, differentiation, and migration, emphasizing the multifaceted role of integrin aV.
The significance of aV integrin is not only linked with normal physiological functions.Dysregulation of aV integrin function has been associated with a variety of pathological conditions, including cancer, metastasis, angiogenesis, and wound healing (115).Additionally, aV integrin is involved in vascular remodeling and fibrosis (117).

i. aVb3
aVb3 is a multifaceted integrin due to its expression on a plethora of cell types and its ability to bind to many extracellular ligands.Through recognizing Arg-Gly-Asp (RGD) motif, aVb3 binds to extracellular matrix proteins such as vitronectin, fibronectin, fibrinogen, and von Willebrand factor (48,118).It can also serve as a receptor of some viruses for their entry into target cells (119).The aVb3-TLRs cooperation has been described in several studies; Plasma membrane TLR4, TLR5 and endosomal TLR3 activated epithelial cells via NF-kB signaling pathway in response to viral and bacterial pathogen-associated molecular pattern molecules (PAMPs) (120).aVb3 further enhanced their NF-kB activation.aVb3 also positively orchestrated TLR2 signaling by facilitating a recruitment of the adaptor MyD88 to TLR2 (121).This mechanism was driven by a physical interaction of both aVb3 and TLR2 with herpes simplex virus (HSV).This leads to NF-kB activation and the production of various mediators including IFNa, IFN-b, IL-2, and IL-10 in response to the viral infection.Another type of aVb3-TLR2 interplay has been attested in a different study, in which aVb3 was shown serve as a co-sensor for bacterial lipopeptide (BLP) to be detected by TLR2 (122).The molecular mechanism mediating TLR2 activation was through the recognition of BLP by vitronectin on human monocytes.The TLR2-aVb3 complex interaction was entirely dissociated following the completion of BLP stimulation.This further confirmed the physical link between aVb3 and TLR2 in recognizing invading pathogens and initiating a synergistic response.The collaboration between aVb3 and TLRs was also described in bacterial infection.In a murine cecal ligation and puncture (CLP)-induced sepsis and in a LPS-stimulated macrophage cell model, aVb3 positively regulated TLR4 signaling in peritoneal macrophages (123).The deficiency of aVb3 attenuated TLR4 activation.This effect appears to be mediated by CD14 expression, as aVb3 deficiency inhibited CD14 expression.The deleterious impact of the aVb3 -CD14-TLR4 crosstalk was caused by the release of a variety of pro-inflammatory cytokines.Therefore, CD61 (b3) KO mice exhibited higher survival rates and were more resistant to septic organ injury.A similar study revealed that thw previous crosstalk was mediated by WNT1 inducible secreted protein 1 (WISP1) (124).Ligation of WISP1 to aVb3 synergistically enhanced TLR4-mediated TNF synthesis in LPS treated peritoneal macrophage.

ii. aVb5
Similar to aVb3, aVb5 serves as a receptor for vitronectin (125).aVb5 mediates phagocytosis of apoptotic cells and promotes angiogenesis and wound healing (126).The interaction of aVb5 with TLR4 during infection was illustrated in a murine two hit-model of CLP and mechanical ventilation (MV)-induced lung injury (127).TLR4 KO mice showed better survival and less lung injury compared to wild type (WT) mice.aVb5 regulated vascular permeability in both ventilator-induced lung injury (VILI) (128) and CLP (129).In line with this knowledge, neutralizing antibodies against aVb5 partially attenuated lung injury.In this model, peritoneal macrophages increased the expression of aVb5 in response to TLR4 activation.The connection between aVb5 and TLR4 contributed to the exacerbations of the CLP-MV lung injury model.
iii. aVb6 aVb6 is expressed mainly on epithelial cells and involved in wound healing (130).Excessive production of aVb6 leads to lung fibrosis and cancer (131).Activation of transforming growth factor-b1 (TGF-b1) represents the key role of aVb6 (131,132).In line with this, influenza infection stimulated TLR3 and further induced aVb6dependent TGF-b1 activation in epithelial cells (132).TLR3-aVb6 crosstalk converged on the RhoA kinase that was activated by TLR3.RhoA kinase was further required to activate TGF-b1 via aVb6.This suggests that the crosstalk was through a signaling pathway rather than a direct physical interaction between TLR3 and aVb6.Blocking aVb6 seemed to have no effect on the viral entry to the epithelial cells or the replication of viral genes.The biological consequences of TGF-b1 activation via aVb6-TLR3 axis were epithelial cell death and accumulation of collagen in mouse lungs, which in turn promoted fibrosis.Another adverse effect of aVb6 during influenza infection of lung epithelium was the suppression of type I IFN response (133).The IFN antiviral response was mainly mediated by endosomal TLR7.aVb6 activated lysosomal autophagy machinery to remove TLR7, leading to the suppression of TLR7-mediated IFN signaling against Influenza infection.Opposite to aVb3, aVb6 seemed to have no physical interaction with TLRs.

Conclusion
Without doubt, TLRs regulate major signaling pathways to modulate the degree of inflammation.While TLRs crosstalk is not exclusively restricted to integrins as complement system has been shown to intercommunicate with TLRs in the host immunity during infection (134), we highlighted ones involving integrins here.As there are a number of signaling pathways to regulate inflammation, it is not surprising that crosstalk system involving integrins has been established to coordinate inflammatory responses as we examined (135).Underhill has proposed several possibilities why the crosstalk has evolved; 1) To provide robust response against invading microbes.2) Compensation against genetic diversity in host population, 3) Multiple receptors can facilitate a more tailored, specific response (136).The idea of "a more tailored, specific response" is very fascinating, because innate immune cells, which usually express TLRs predominantly, are rather considered promiscuous and relatively non-specific compared to adaptive immunity.Further understanding the role of crosstalks between TLRs and integrins would allow us to understand very complex system that innate immunity has developed and intervene if indicated.

FIGURE 3 aMb2
FIGURE 3    aMb2 and TLR4 crosstalk The crosstalk between aMb2 and TLR4 is shown.Inside-out signal: LPS binding to TLR4 induces the activation of many molecules including PI3K.PI3K facilitates the activation of aMb2 intracellular adaptor proteins, therefore aMb2 itself.Outside-in signal: Activated aMb2 communicates with Src/Syk, which facilitates the degradation of MyD88 and TRIF.This will attenuate TLR4 activation signal.Of note, Syk is typically associated with other receptors like C-type lectin receptors (CLRs).

TABLE 1
List of TLRs, their location and ligands.